Optimization Of Somatic Embryogenesis System And Constructing Genetic System Of Torreya Grandis 'Merrillii'

Torreya grandis‘Merrillii'is Chinese Torreya grandis belonging to belongs toTaxales,Taxaceae,Torreya.And It is the evergreen tree.The Torreya grandis‘Merrillii'is a native species of China,which is mainly grown in humid areas in the south of China,and a rare economic tree in the world.The seeds of Torreya grandis‘Merrillii'are full of rich nutrition and they can be used for health protection medicine and comprehensive development.In the East Asian countries,the wood of Torreya grandis‘Merrillii'trees are the advanced material for chessboard.Recent years,there are some reports which are about somatic embryos induction and plant regeneration and organ culture in vitro regenerated plants.However,there is barely thesis about variety improvement and establishment of genetic transformation system.This paper is aimed at optimizating somatic embryogenesis systemand illuminate the main factor of genetic transformation for constructing stable and comprehensive system of excellent somatic embryos expending propagation for Torreya grandis‘Merrillii'and genetic transformation.It may provide the important technology for functional validation of important genes and selection of new varieties.At the same time,it can support the healthy and sustainable Zhejiang's Torreya grandis‘Merrillii'industry.This experiment is aimed at optimizing the system of the condition of inducing and propagating somatic embryos for Torreya grandis‘Merrillii'.And then transfer the empty vector pCAMBIA1300 with green fluorescence protein?GFP?into the somatic embryos via agrobacteria for finding the best condition.The main result is following:1.On the basis of the system of regeneration of somatic embryos of Torreya grandis‘Merrillii',the conditions of somatic embryo induction culture were optimized.It shows that t he induction rate of callus reached 86.7%in the culture media of 1/2SH+NAA0.1mg/L+Gl n 0.5mg/L+AC 2g/L+sucrose 3%in four weeks.At the same time,it was found that the ad dition of Gln could promote induction rate and germination rate of callus and germination rate o f somatic embryo in the early period.2.SH medium was used as the basal medium,adding different concentrations of inorganic salt?1/2SH?SH?,different concentrations NAA?0.1mg/L,0.05mg/L?,different concentrations of Gln and hydrolytic protein?CH?,and figure out which is the best culture condition.Finally,the results show that the propagation of somatic embryos can reach at the peak of 7.4 times,and the number of somatic embryogenesis is 76 as the highest when the medium is 1/2SH+NAA0.1 mg/L+Gln0.5mg/L+AC 2g/L+sources3%.And then,primordial embryos can be cultured in the medium of 1/2SH+ABA10mg/L+PEG60g/L+AC 2g/L+sources3%.It can make rod like embryos grow into cotyledons continually.3.in order to construct the system of genetic transformation ofTorreya grandis‘Merrillii'.We tested if the different growth periods of immature embryoswould effect the efficiency of transformation.The result showed that it was not the key element.What we found in this study was that differentmaterial,infected timeandconcentration had impacted a significant influence on genetic transformation efficiency.Immature embryos was the best material.And infected timeandconcentration effects genetic transformation efficiency negatively.Under condition of infected10min,OD₆₀₀=0.5,it was perfect to con-culture for 3 days and to move the material on the medium of 1/2SH+NAA0.1mg/L+Hygromycin80mg/L+Carbenicillin300mg/L at the23?for 2weeks.And then,the material were also needed to move to the medium of 1/2SH+NAA0.1mg/L+Hyg100mg/L+Car300mg/L for 4 weeks.In this situation,the expression of GFP can reach at 98%.The positive somatic embryoss were used as the material for PCR.Finally,the results proved that the positive tissue containing GFP signal.